1. Field of the Invention
The present invention relates to novel immortalized cell lines of retinal origin (retinal endothelial and retinal pigmentary epithelial origin) which are capable of being implanted in the retina and of conveying a substance of therapeutic interest into the eye and the central nervous system. Such lines can also serve as a model for studying the blood/central nervous system interfaces.
2. Description of the Background
Both the blood-brain barrier and the blood-retina barrier are important in controlling the passage of substances to and from the neural parenchyma, especially in the maintenance of haemostasis.
In the retina, the blood-retina barrier comprises two different types of cells which are anatomically separate. The retinal vascular endothelium, which supplies the anterior portion of the retina, is currently considered to have an identical structure to the cerebral endothelium (Towler et al., J. Physiol., 1994, 480, 10-11P), whereas the cells of the retinal pigmentary epithelium cover the permeable vessels of the choroidal circulation and form the posterior barrier by means of their tight apical junctions; they are consequently similar to the tight junction epithelial cells of the choroid plexus.
The cerebral and retinal endothelia are of a different nature to the peripheral endothelium and do not serve only to express tight junctions and form a physical barrier. Other properties of the endothelium contribute to the specialized nature of this barrier, particular properties being the distribution and expression of substances such as the glucose transporter (GLUT-1), the transferrin receptor and P-glycoprotein (Pgp), the expression product of the drug resistance gene.
The cerebral and retinal endothelia also differ from the peripheral endothelium in their permeability to the circulating leukocytes.
The in vitro study of the molecular mechanisms of the induction of the endothelial phenotype has the disadvantage of being dependent on the availability, in primary cell cultures, of endothelial cells of cerebral or retinal vessels or pigmentary epithelial cells of the retina.
As far as in vivo transfer is concerned, the use of primary nerve tissues of foetal origin for cellular transplantation in human therapy gives rise to numerous ethical and practical problems; one alternative to this problem is to use primary cell lines of neural origin (for example neurons, glial cells, astrocytes) or non-neural cell cell lines (for example fibroblasts, myoblasts, chromaffin cells of the adrenal medulla, hepatocytes). Although the cell lines of the adrenal medulla, fibroblasts or myoblasts can actually release active substances in vivo, they are not normally present in the central nervous system, but can modify the normal function of the nervous system and cause a rejection reaction.
Because of the heterogeneity of the endothelial cells of different tissues, influenced by the environment of these cells, it was important to be able to have cells adapted to the retinal environment in order to have tools permitting a good morphological and physiological integration of the cells when they are implanted or grafted.
Consequently, the Inventors set themselves the task of providing immortalized cell lines derived from primary cultures of the endothelium of the blood-retina barrier and the retinal pigmentary epithelium of mammals, especially rodents and more particularly rats, which cell lines are better capable of meeting practical needs, especially in that all the lines obtained are stable and have most of the characteristics of the differentiated cells.